Streptomyces species are attractive hosts for the secretion of heterologous proteins because they are non-pathogenic, efficiently secrete proteins directly into the media, and their cultivation on an industrial scale is well understood. Currently, S. lividans is being utilized as the prototype host for the export of heterologous proteins from Streptomyces since detailed procedures have been established for its manipulation via classical genetic or recombinant DNA technologies [Genetic Manipulations of Streptomyces, Hopwood et al., Eds., The John Innes Foundation, 1985]. Indeed, S. lividans has been used as a host to direct the export of human interleukin 1.beta., E. coli .beta.-galactosidase, E. coli alkaline phosphatase, human tumor necrosis factor, and human interferon .alpha.-1 [Bender et al., (1990) Gene, vol. 86, pp: 227-232; Lichenstein et al., (1988) J. Bact., vol 170, pp: 3924-3929; Noack et al., (1988) Gene, vol. 68, pp: 53-62; Chang et al. in T. Okami et al., (1988) Biology of Actinomycetes, Japan Soc. Press, Tokyo, pp: 103-107].
Of particular concern in the use of S. lividans as a host for the secretion of heterologous proteins is degradation of the exported products by endogenous proteolytic activities. Numerous proteases have been shown to be secreted by S. lividans and these proteases could potentially damage the desired product [Aretz et al., (1989) FEMS Microbiology Letters, vol. 65, pp: 31-36]. To date, there have been no reports describing the cloning of a protease gene from S. lividans. The isolation of such a gene could prove invaluable in that the gene encoding the protease could be modified using recombinant DNA techniques to eliminate the proteolytic activity of the protein. The mutated gene for this deactivated protease would then be substituted for the wild-type protease gene by chromosomal integration. The resulting S. lividans strain would be protease-deficient and better suited for the secretion of heterologous protein products.
Isolation of a protease gene could also be useful in the design of vectors directing the expression and secretion of heterologous proteins from S. lividans. As proteins secreted from S. lividans are generally first synthesized with a leader peptide, it is likely that a secreted endogenous protease from S. lividans would contain such a leader or signal sequence. Thus, this leader peptide sequence could be used to direct the export of heterologous proteins. Additionally, regulatory sequences for the protease might also prove useful in enhancing the expression of the heterologous protein.
Use of an endogenous signal sequence to direct secretion of a foreign protein would facilitate the proper processing of the signal peptide by the S. lividans export machinery. Signal sequences isolated from other Streptomyces species, or isolated from other bacterial genera, may not function efficiently, or at all, in S. lividans. To date, the .beta.-galactosidase signal peptide has been the only signal peptide isolated from S. lividans that has been used to direct the export of heterologous proteins from this organism [Lichenstein et al, (1988) supra].
The isolation of a novel protease gene from S. lividans may also define a protease with a unique activity which could prove commercially useful. For example, it is known that Streptomyces proteases, as well as those from other microbial species, are used in the food (protein liquefaction, milk clotting, meat tenderizing), pharmaceutical (fibrinolytic, thrombolytic), and tanning industries or as detergent additives. They can also be used in the research environment in the structural determination of proteins or in the removal of proteinaceous material during biopreparations. Examples of Streptomyces protease preparations that are commercially useful include PRONASE.TM. (S. griseus) and FRADIASE.TM. (S. fradiae). Other Streptomyces proteases have been isolated and together they comprise a large pool of proteases having various substrate specificities [Goodfellow et al. (Eds.), Actinomycetes in Biotechnology, 1988, Academic Press, pp: 246-250]. The cloning of such protease genes from S. lividans would allow the amplification of protease production such that sufficient quantities could be generated for any commercial application in which the protease could be utilized.